The long-term objectives of this proposal are to understand the molecular and cellular mechanisms of auditory mechanosensation. These goals will be achieved by using a new genetic model system for hearing in Drosophila. The public availability of almost the entire genome sequence, together with the genetic, developmental and molecular tools for manipulating Drosophila make this a very powerful model. Hearing in insects is mediated by chordotonal organs, which are related to vertebrate auditory and vestibular hair cells because they are developmentally specified by homologs of the same gene, atonal. The first approach will be to identify mutations that specifically disrupt chordotonal organ function, to clone the corresponding genes and to elucidate the cellular location and molecular function of their gene products. Mutations in three genes, beethoven, smetana and touch-insensitive-larva-B will be subjected to this analysis. The second approach will make use of enhancer trap strains, whose engineered transposon inserts express a reporter gene specifically in chordotonal organs. Four enhancer trap strains identified by this criterion will be used as starting points to clone the flanking sequences to identify candidate chordotonal-specific genes. If the transposon does not disrupt the gene, imprecise excision derivatives will be generated as a way to introduce mutations in the gene. The transposons therefore act not only as reporters, but also as molecular tags and as a mutagen. The third approach will use known human genes associated with deafness as a starting point to identify Drosophila homologs and then to use reverse genetics to identify mutations in these genes to test for function. Methods for this reverse genetic approach will include characterization of nearby transposon insertions or their imprecise excision derivatives as well as recently described gene replacement strategies. Identifying auditory genes by any of these approaches and elucidating the molecular roles of their products will provide very important insight into the fundamental but poorly understood process of mechanosensation.